2. Patients and Methods.
Study participants. We obtained the demographic and clinical
data of 482 COVID-19 patients (age range 24-95 years) who required
admission in the intensive care unit (ICU). The study was approved by
the Ethics Committee of Principado de Asturias (Oviedo, Spain; approval
id project ISCIII-PI21/00971), and all the patients (or their next of
kin) gave informed consent to participate in the study. These patients
were hospitalised between March-2020 and December-2021, a period with
four SARS-CoV-2 pandemic waves in our community. We did not determine
the SARS-CoV-2 variant in all the patients, but the study period was
characterised by the Wuhan (pandemic waves 1-2), alpha (wave 3) and
delta (wave 4) variants.
The inclusion criteria was a severe pneumonia in need for ICU admission
with SARS-CoV-2 confirmed by PCR test, and exclusion criteria were age
<18, respiratory failure due to condition other than COVID-19,
or refusal to participate. These patients were followed till disease
remission with hospital discharge or death. Body-mass index (BMI) and
preexisting cardiovascular comorbidities (hypertension, diabetes,
hypercholesterolemia) were obtained from the clinical history at ICU
admission. Based on previous reports we compared COVID-19 patients aged
≤60 and >60 years [Nakanishi et al., 2021]. At
the time of inclusion, none of the patients had a recorded diagnostic of
mitochondrial disease.
All the participants were of European ancestry from the region of
Asturias (Northern Spain, total population 1 million). The controls
(N=363) were individuals from the general population recruited with the
main purpose of defining the DNA variant frequencies. These controls
were not serologicaly studied (presence of anti-SARS-CoV-2 antibodies)
to exclude previous asymptomatic infection. In order to exclude the
posibility of age-bias in the genotype frequencies we compared patients
and controls within the same age-range.
Genetic analysis. Patients and controls were studied for mtDNA
single nucleotide polymorphisms (SNPs) C7028T and C16223T by Sanger
sequencing of PCR fragments. The 7028 polymorphism differentiates the
major European haplogroup H (7028C) from no-H (7028T), while 16223
differentiates the ancestral African macro-haplogroup N (16223T) from
the out-of-Africa macro-haplogroup R (16223C)
(www.mitomap.org) . This method
allowed to determine the presence of heteroplasmy at the 7028 and 16223
nucleotides.
To define the degree of length heteroplasmy at the 16184-16193 poly C
tract we performed a fluorescent capillary electrophoresis of PCR
fragments generated with primers FAM-5´CTGCCAGCCACCATGAATATTGTACGG and
5´GTGGCTTTG GAGTTGCAGTTGATGTGTGA (annealing at 65ºC). The forward primer
was labelled with FAM and fragments were thus visualised as fluorescent
peaks after capillary electrophoresis (Figure 1A ). Homoplasmic
fragments corresponded to single peaks while the presence of additional
peaks was considered as heteroplasmy. We also characterised the degree
of heteroplasmy at the CA-repeat between nucleotides 514-523 by
fluorescent capillary electrophoresis of PCR fragments with primers
fam-5´ CACTTTTAACAGTCACCCCCCAACTAAC and 5´TTCGGGGTATGGGGTTA GCAGCG
(annealing at 65ºC) (Figure 1B ). To discrimitate true from PCR
stutter peaks (characteristic of amplicons containing nucleotide
repeats) we condidered heteroplasmy when the additional peaks had a
heigh >10% relative to the main fluorescent peak
[Walsh et al., 2016] . The accuracy of this method was
validated by sequencing PCR fragments with different degrees of
heteroplasmy (suppl figure ).
Statistical analysis. All the values were collected in an
excell file. The statistical analysis was performed with the R-software
(www.r-project.org). Logistic
regression (linear generalized model, LGM) was used to compare mean
values and frequencies between the groups.